The study was approved by the Ethical Committee of Osaka University Graduate School of Medicine. Written informed consent was obtained from all of them. All healthy volunteers were negative for HCV, RAD001 clinical trial hepatitis B virus (HBV), and human immunodeficiency virus (HIV) and had no apparent history of liver, autoimmune, or malignant diseases. The specifications

of all antibodies used for FACS or cell sorting TLR-specific synthetic agonists, pharmacological reagents, and inhibitory peptides are listed in the Supporting Materials. We collected 400 mL of blood from each healthy volunteer and processed them for PBMCs. Noncancerous liver tissues were obtained from patients who underwent resection of liver tumors (Supporting Table 1). For the collection of intrahepatic lymphocytes (IHLs), liver AZD9668 tissues were washed thoroughly with phosphate-buffered saline to remove the peripheral blood adhering to the tissue and ground gently. After Lin-negative (CD3−, CD14−, CD19−, and CD56−) cells were obtained by the MACS system, each DC subset with the defined phenotype was sorted separately under FACS Aria (BD). The purity was more than 98%, as assessed by FACS Canto II (BD). Sorted

DCs were cultured at 2.5 × 104/well on 96-well culture plates. Tissue specimens were obtained from surgical resections of noncancerous liver from the patients as described above. Briefly, the 5-mm sections were incubated with the following antibodies: mouse biotinylated antihuman BDCA3 antibody (Miltenyi-Biotec), and mouse antihuman CLEC9A antibody (Biolegend) and subsequently with secondary goat antirabbit Alexa Fluor488 or goat antimouse Alexa Fluor594 (Invitrogen, Molecular Probes) antibodies. Cell nuclei were counterstained with Dapi-Fluoromount-GTM (Southern 3-mercaptopyruvate sulfurtransferase Biotech, Birmingham, AL). The stained tissues were analyzed by fluorescence microscopy (Model BZ-9000; Keyence, Osaka, Japan). The in vitro transcribed RNA of the JFH-1 strain of HCV was introduced into FT3-7 cells12 or Huh7.5.1 cells. The stocks of HCVcc were generated by concentration of the medium from JFH-1-infected FT3-7 cells. The virus

titers were determined by focus forming assay.13 The control medium was generated by concentration of the medium from HCV-uninfected FT3-7 cells. Infectious JEVs were generated from the expression plasmid (pMWJEATG1) as reported.14 HSV (KOS) was a generous gift from Dr. K. Ueda (Osaka University). Huh7.5.1 cells transduced with HCV JFH-1 strain was used for the coculture with DCs. The transcripts of ISGs in Huh7.5.1 were examined by reverse-transcription polymerase chain reaction (RT-PCR) methods using gene-specific primers and probes (Applied Biosystems, Foster City, CA). IL-28B/IFN-λ3 was quantified by a newly developed chemiluminescence enzyme immunoassay (CLEIA) system.15 IL-29/IFN-λ1, IL-28A/IFN-λ2, and IFN-β were assayed by commercially available enzyme-linked immunosorbent assay (ELISA) kits (eBioscience, R&D, and PBL, respectively).